The Glucocorticoid Resistance Syndrome. Two Cases of a Novel Pathogenic Variant in the Glucocorticoid Receptor Gene

Abstract Glucocorticoid resistance syndrome is a rare genetic condition characterized by generalized or partial target-tissue insensitivity to glucocorticoids and a consequent hyperactivation of the hypothalamic-pituitary-adrenal axis. Clinical manifestations may include mineralocorticoid and/or androgen excess without manifestations of Cushing syndrome. At a cellular level, glucocorticoid actions are mediated by the nuclear glucocorticoid receptor encoded by the NR3C1 gene. To date, only 33 glucocorticoid receptor loss-of-function pathogenic variants have been associated with glucocorticoid resistance syndrome. The NR3C1 gene has 2 known disease-causing mechanisms: haploinsufficiency and negative dominance. We describe a mother and her son with a mild hyperandrogenic phenotype and a novel genetic variant of the NR3C1 gene predicting a truncated protein and causing glucocorticoid resistance syndrome. To date, no accurate genotype-phenotype correlation has been found.


Introduction
Glucocorticoids are steroid hormones synthesized in the zona fasciculata of the adrenal cortex and act as the end products of the hypothalamic-pituitary-adrenal axis.Glucocorticoids have a wide range of physiological processes associated with growth, inflammatory processes, tolerance to stress, carbohydrate-protein-fat and bone metabolism, and sexual development.They work by binding to the human glucocorticoid receptor (hGR), a ubiquitously expressed protein located in the nuclear region of target cells.Both synthetic and natural glucocorticoids can bind to the hGR [1].
GRS is an extremely rare endocrine disorder that affects tissues expressing the hGR and is characterized by a generalized or partial decreased sensitivity to glucocorticoids due to a defective hGR in both the hypothalamus and pituitary gland that results in an impaired cortisol negative feedback loop, driving hypersecretion of corticotropin-releasing hormone, arginine vasopressin, and ACTH.The increased plasma ACTH concentrations lead to stimulation of the adrenal cortex with release of cortisol, adrenal androgens (androstenedione, dehydroepiandrosterone), mineralocorticoid steroids (corticosterone and deoxycorticosterone), and adrenal hyperplasia.
Patients with GRS may have biochemical hypercortisolism without Cushing disease symptoms.The clinical phenotype is diverse, ranging from asymptomatic cases to severe mineralocorticoid (hypertension, hypokalaemia, and alkalosis) and/or androgen excess (hirsutism, acne, male-pattern hair loss, precocious puberty, amenorrhoea or hypo fertility, and menstrual irregularities in females and oligospermia in males).Hypercortisolism may be the cause of patient persistent fatigue while increased corticotropin-releasing hormone and arginine vasopressin secretion may lead to anxiety and sadness in these patients.
Diagnostic evaluation of the GRS includes a complete medical history with an emphasis on the absence of symptoms and signs of hypercortisolism, determination of morning serum cortisol under fasting conditions and after dexamethasone administration, 24-hour urinary free cortisol (UFC), plasma ACTH, serum aldosterone, plasma renin activity, and androgens.The NR3C1 sequencing is required for diagnostic confirmation.

Case 1
A 49-year-old female was evaluated at the endocrine department because of hirsutism, childhood acne, and irregular menstrual cycles.She had conceived through in vitro fertilization and was being treated for anxiety.She had gone through several rounds of hair removal sessions.No skin atrophy, buffalo hump, purple striae, myopathy, or truncal obesity were present.Her body mass index was 22.6 kg/ m2, and blood pressure, potassium levels, and glucose homeostasis were normal.

Case 2
We also analyzed her 17-year-old son, who presented precocious puberty with severe hirsutism, acne, short stature (height 165 cm, body mass index 20 kg/m2), and advanced bone maturation.His blood pressure was 128/70 mmHg.Laboratory findings (Table 1) also revealed increased ACTH and 24-hour UFC and resistance of the hypothalamicpituitary-adrenal axis to low-dose dexamethasone suppression.Genetic analysis revealed he also was a carrier of the mother's variant.
Some possible differential diagnosis may include: • Mild forms of Cushing syndrome, in which hypercortisolism is accompanied by normal or mildly elevated ACTH concentrations, preserved circadian pattern of ACTH and cortisol secretion, and lack of cortisol supression by dexamethasone.• Pseudocushing states, such as genealized anxiety disorder or melancholic depression.• Conditions associated with elevated serum concentrations of cortisol-binding globulin.
• Other causes of hyperandrogenism or virilization such as polycystic ovarian syndrome in females or congenital adrenal hypeplasia.

Diagnostic assessment
Hormone laboratory findings are shown in

Treatment
Treatment aims to reduce excessive endogenous ACTH secretion, especially in patients with significant hGR action impairment, by administering increasing doses of synthetic glucocorticoids.

Outcome and Follow-up
Following genetic counselling and written informed consent, polymerase chain reaction amplification and Sanger sequencing were used to analyze the NR3C1 gene, and the c.2024-1G > T variant was found in heterozygosis.This variant is located at the end of intron 7 (Fig. 1) and predicts the skipping of the first 8 coding nucleotides of exon 8 (Fig. 2).

Discussion
We report a second splice-site variant (c.2024-1G > T) of the NR3C1 gene.In this case, this change occurs in the nuclear localization signal 2, predicting a protein frameshift from codon 675, p.(Val675Glyfs*10) and, as a consequence, a premature terminal codon [6].
The NR3C1 gene has known 2 disease-causing mechanisms: haploinsufficiency and negative dominance.Haploinsufficiency can be caused by changes that result in truncated proteins but also by missense variants, and negative dominance is mainly caused by missense changes.Taking both mechanisms into account, it would be noteworthy to check if variants predicted to produce a frameshift correlate with a mild clinical phenotype (as observed in our case) because there is practically no interference with the normal product.When more variants predicting frameshift or missense changes are described, we could envisage this possible genotype-phenotype correlation.

Learning Points
• GRS is a rare endocrine disorder due to mutations in the hGR encoded by the NR3C1 gene.• The clinical spectrum of GRS is broad, ranging from most severe to mild features of mineralocorticoid and/or androgen excess.• Laboratory findings present normal or elevated plasma ACTH concentrations with increased 24-hour UFC excretion and inadequate suppression of morning serum cortisol to low-dose dexamethasone suppression test.
• To date, 33 NR3C1 loss-of-function pathogenic variants have been associated with GRS.